Lung gamma delta T cells in tumor-free and mammary tumor-bearing mice
Ontology highlight
ABSTRACT: Gamma delta T cells were sorted from the lungs of 4 female FVB/n mice (labeled wild-type [WT] 1-4) and 4 female mammary tumour-bearing K14-Cre;Brca1F/F;Trp53F/F (KB1P) mice (labeled Tumour 1-4).The sorted cells were loaded onto the Chromium Single Cell 30 Chip Kit v2 (10xGenomics) to generate libraries for scRNAseq, following the manufacturer’s instructions. The sequencing-ready library was cleaned up with SPRIselect beads (Beckman Coulter, High Wycombe, UK). Quality control of the library was performed prior to sequencing (Qubit, Bioanalyzer, qPCR). Illumina sequencing was performed using NovaSeq S1 by Edinburgh Genomics (University of Edinburgh).
Project description:Single-cell RNA-seq and TCR-seq were conducted using coding RNA of single cells isolated from mouse lung MNCs with or without 7DW8-5 according to the manufacturer’s instructions. In brief, single cells from the mouse lung MNCs were incubated with mouse Fc block and then stained by anti-mouse CD45+ antibody and DAPI. Sorted viable CD45+DAPI- single cells were loaded into 10x genomics ChromiumTM controller (samples were split into 4 lanes, Saline1 and Saline2 without 7DW8-5 treatment, and 7DW1 and 7DW2 with 7DW8-5 treatment) to make nanoliter-scale droplets with uniquely barcoded 5’ gel beads called GEMs. After GEM-RT and the following some cDNA amplification steps, cDNAs derived from cellular mRNA were pooled for downstream processing and library preparation according to the manufacturer’s instructions. The 5’ transcript library and TCR library were sequenced with Illumina Novaseq. Fastq files from RNA-seq and TCR-seq can be processed through cellranger and vdjranger by 10xgenomics. The datasets include the data of the samples Saline1, Saline2, 7DW1, and 7DW2.
Project description:RNAseq analysis between cell types. RNA extracted using Qiagen mini total RNA isolation protocols (Qiagen, Germany). Samples were treated with DNase (Ambion), and sample integrity verified on the Agilent Bioanalyser with the RNA Nano chip. Illumina Tru-seq paired end strand specific sequencing (Illumina, USA) was carried out by on a NextSeq-550 sequencer (Edinburgh Clinical Research Facility, Western General Hospital, Edinburgh, Scotland, UK). 500ng of Total RNA underwent ribosomal RNA depletion (rRNA) prior to purification, fragmentation, random hexamer cDNA generation and purification with AMPure XP beads (Beckman-Coulter, USA). Multiple indexing adapters were ligated to ds cDNA with subsequent hybridisation onto flow cells, and DNA fragment enrichment by 15 cycle PCR for sequencing. Completed libraries were quantified by qPCR using the KAPA Illumina Library Quantification Kit (Illumina, USA) before multiplexing in two equimolar pools and running on two flow cells on the Illumina NextSeq 550. The resulting FastQ files were mapped to the reference genome (mm9) using the Tophat alignment tool (V1) on Illumina Basespace software and reads per kilobase per million (RPKM) scores calculated.
Project description:RNAseq analysis between cell types. RNA extracted using Qiagen mini total RNA isolation protocols (Qiagen, Germany). Samples were treated with DNase (Ambion), and sample integrity verified on the Agilent Bioanalyser with the RNA Nano chip. Illumina Tru-seq paired end strand specific sequencing (Illumina, USA) was carried out by on a NextSeq-550 sequencer (Edinburgh Clinical Research Facility, Western General Hospital, Edinburgh, Scotland, UK). 500ng of Total RNA underwent ribosomal RNA depletion (rRNA) prior to purification, fragmentation, random hexamer cDNA generation and purification with AMPure XP beads (Beckman-Coulter, USA). Multiple indexing adapters were ligated to ds cDNA with subsequent hybridisation onto flow cells, and DNA fragment enrichment by 15 cycle PCR for sequencing. Completed libraries were quantified by qPCR using the KAPA Illumina Library Quantification Kit (Illumina, USA) before multiplexing in two equimolar pools and running on two flow cells on the Illumina NextSeq 550. The resulting FastQ files were mapped to the reference genome (mm9) using the Tophat alignment tool (V1) on Illumina Basespace software and reads per kilobase per million (RPKM) scores calculated.
Project description:RNAseq analysis between control (normoxia) and hypoxic mice +/- bacterial infection. Murine peripheral blood leukocytes (1x10^6/condition) were lysed and RNA extracted using Qiagen mini total RNA isolation protocols (Qiagen, Germany). Samples were treated with DNase (Ambion), and sample integrity verified on the Agilent Bioanalyser with the RNA Nano chip. Illumina Tru-seq paired end strand specific sequencing (Illumina, USA) was carried out by on a NextSeq-550 sequencer (Edinburgh Clinical Research Facility, Western General Hospital, Edinburgh, Scotland, UK). 500ng of Total RNA underwent ribosomal RNA depletion (rRNA) prior to purification, fragmentation, random hexamer cDNA generation and purification with AMPure XP beads (Beckman-Coulter, USA). Multiple indexing adapters were ligated to ds cDNA with subsequent hybridisation onto flow cells, and DNA fragment enrichment by 15 cycle PCR for sequencing. Completed libraries were quantified by qPCR using the KAPA Illumina Library Quantification Kit (Illumina, USA) before multiplexing in two equimolar pools and running on two flow cells on the Illumina NextSeq 550. The resulting FastQ files were mapped to the reference genome (mm9) using the Tophat alignment tool (V1) on Illumina Basespace software and reads per kilobase per million (RPKM) scores calculated.
Project description:Cancer is a heterogeneous disease, where multiple, phenotypically distinct subpopulations co-exist. Tumour evolution is a result of a complex interplay of genetic and epigenetic factors. To predict the molecular drivers of distinct cancer responses, we apply single-cell lineage tracing (scRNA-Seq of barcoded cells) on a triple-negative breast cancer model. SUM159PT cells infected with a lentiviral barcode library (Perturb-seq Library) were sorted according to the presence of BFP signal, treated or not with paclitaxel (PTX), multiplexed with MULTI-Seq protocol, and then processed by scRNA-Seq.
Project description:Cancer is a heterogeneous disease, where multiple, phenotypically distinct subpopulations co-exist. Tumour evolution is a result of a complex interplay of genetic and epigenetic factors. To predict the molecular drivers of distinct cancer responses, we apply single-cell lineage tracing (scRNA-Seq of barcoded cells) on a triple-negative breast cancer model. SUM159PT cells infected with a lentiviral barcode library (Perturb-seq Library) were sorted according to the presence of BFP signal, treated or not with paclitaxel (PTX), and then processed by scRNA-Seq or Multiome.
Project description:Mice implanted with CT26 tumour cells were divided into two experimental groups (vehicle and ceralasertib) with the aim of assessing changes in intra-tumour T cells (CD3+) after 7 days of treatment with ceralasertib, and 7 days off. At the end of the treatment course, tumour tissue was isolated, chopped and enzymatically digested to generate a single-cell suspension. Cells sorted on CD3 were then processed using Chromium Next GEM Single Cell 5' Kit v2.
Project description:Chimeric mouse embryos were generated as a control experiment to understand the contribution of host vs. injected cells to the developing embryo. Embryos were generated by blastocyst injection of tdTomato-labelled mouse embryonic stem cells into wild type embryos. After blastocyst harvest, cells were flow-sorted before 10X Genomics library preparation and single-cell RNA-sequencing.
Project description:Chimeric embryos were generated to investigate the effect of Tal1 knockout in mouse embryos by single-cell RNA-sequencing. Tal1 is an essential transcription factor for the formation of the embryonic blood. Embryo chimerism permits the analysis of the effects of Tal1 knockout without the confounding effects of the absence of embryonic blood, which results in global developmental failures. Embryos were generated by blastocyst injection of tdTomato-labelled, Tal1-/- mouse embryonic stem cells into wild type embryos. After blastocyst harvest, cells were flow-sorted before 10X Genomics library preparation and single-cell RNA-sequencing.